Mutation of Angiogenesis Inhibitor TK1-2 to Avoid Antigenicity In Vivo

Lee Sang-Bae; Kim Hyun-Kyung; Oh Ho-Kyun; Hong Yong-Kil; Joe Young-Ae

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자료유형: 학술저널

저자정보: Lee Sang-Bae (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) Kim Hyun-Kyung (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) Oh Ho-Kyun (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) Hong Yong-Kil (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) Joe Young-Ae (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea)

저널정보: 한국응용약물학회 The journal of applied pharmacology : the official journal of the Korean Society of Applied Pharmacology The journal of applied pharmacology : the official journal of the Korean Society of Applied Pharmacology 제14권 제1호

발행연도: 2006.1

수록면: 30 - 35 (6page)

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Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.

#tissue-type plasminogen activator #kringle doamin #angiogenesis #N-glycosylation #site-directed mutagenesis #Pichia pastoris

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